Project summary Lymphocyte entry and migration within tissues play a critical role in immune surveillance and execution of effector functions, as well as in diseases such as autoimmunity. To properly localize within tissues and interact with antigen presenting cells (APCs) T cells extravasate across the vascular endothelial cell wall and follow environmental cues present in the tissues. During these processes, T cells change shape and extend membrane protrusions such as lamellipodia and filopodia, which require actin polymerization. However, little is known about the specific effectors of actin network remodeling necessary for T cell extravasation and to efficiently survey APCs for antigen. Our preliminary data show that the cytoskeletal effector proteins of the Ena/Vasp family mediate T cell entry into tissues, including the brain during autoimmune inflammation, revealing a specific and previously unknown role for Ena/Vasp proteins in T cell extravasation. T cells undergo a migratory `pathfinding' process following environmental cues to find permissive extravasation sites along vascular walls and to properly localize within tissues during interstitial migration. Filopodia can mediate sensing of environmental stimuli and have been suggested to play a role in T cell extravasation. In neurons and other cell types, Ena/Vasp proteins can play a role in filopodia formation. However, the specific functions of filopodia in promoting T cell extravasation, interstitial motility, and T cell-APC interactions remain unclear. Our goal is to determine the mechanisms by which Ena/Vasp proteins mediate T cell trafficking and interactions with APCs during immune surveillance as well as autoimmune disease. We hypothesize that Ena/Vasp-mediated actin network remodeling and generation of filopodia are required for T cell pathfinding during extravasation, interstitial motility, and surveying of APCs. To address this key gap in the understanding of the mechanisms by which Ena/Vasp proteins regulate pathfinding during lymphocyte trafficking and immune surveillance, we propose three aims: Aim 1: Determine the role of Ena/Vasp proteins in T cell extravasation. Aim 2: Determine the role of Ena/Vasp proteins in T cell immune surveillance and activation. Aim 3: Determine the role of Ena/Vasp proteins in self-reactive T cell trafficking to the Central Nervous System and in autoimmune responses. To accomplish our aims, we will use a multi-faceted approach including genetic, biochemical and cutting-edge imaging techniques (such as 2-photon microscopy and super-resolution microscopy). This will allow us to investigate the mechanisms by which Ena/Vasp proteins regulate T cell extravasation and cell-cell interactions in model systems as well as in physiological environments in vivo. Our work will determine how Ena/Vasp-mediated actin network remodeling regulates T cell migration, pathfinding and activation during extravasation, immune surveillance and autoimmune disease. We will also determine if the Ena/Vasp pathway is a potential target to inhibit T cell activation and trafficking in disease settings such as autoimmunity.